Process for prophylaxis and therapy of thrombotic processes employing heparin having particular average molecular weights

ABSTRACT

A process for prophylaxis and therapy of thrombotic processes includes preparing heparin having an average molecular weight ranging from 10 to 11.5 kd; formulating a pharmaceutical preparation which includes the heparin; and employing the pharmaceutical preparation for prophylaxis and therapy of thrombotic processes. Preferably the heparin has an average molecular weight of 10.5 kd.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to a novel medium molecularweight heparin, its preparation and use.

2. Description of the Related Art

Heparin was discovered by MacLean in 1916 and has been employed inmedicine for more than 60 years. Its main field of application isantithrombotic prophylaxis and therapy. Meanwhile, heparin is used inmedicine in two different forms. The first form is unfractionatedheparin (UFH) which is recovered on an industrial scale from the lungs,livers or intestinal mucosae of cattle and pigs, and is obtained afterproteolysis, separation of undesirable accompanying materials, such asfat and proteins, and bleaching. The second form is low molecular weightheparins (LMWH) which are obtained by the depolymerization of UFH.

The essential difference in the application of these two heparinsresides in their different bioavailabilities. After subcutaneousinjection of UFH, this is about 10–20% of the administered dose, whilethe bioavailability of LMWH is around 90% (R. J. Kandotas, HeparinPharmacokinetics and Pharmacodynamics, Clinical Pharmacokinetics, 22(5):359–374, 1992).

In the following Table 1, the essential characteristics of UFH and LMWHare listed for comparison (J. Hirsh and M. N. Levine, “Low molecularweight heparine”, Blood 79(1): 1–17, 1992).

TABLE 1 UFH LMWH Molecular weight (in Dalton) 3000–30,000 2000–8000Average molecular weight (in Dalton) 13,000 5000 Anti-Xa/anti-IIaactivity 1:1 2:1–5:1 Neutralization by PF 4* strong weak Binding toproteins high low Binding to endothelial cells yes weak Dose-dependentclearance yes no Bioavailability (s.c.) 10–25% about 90% Half-lives(t_(½)) 1 h (i.v.) 2 h (i.v.) 1–2 h (s.c.) 3–6 h (s.c.) Eliminationbiophasic renal Laboratory controls required not required *PF 4 = aplasma protein called platelet factor 4

In clinical applications, UFH and LMWH are essentially employed for theprophylaxis and therapy of thrombo-embolic diseases, LMWH only recentlyhaving found increased use. All in all, the peri- and post-operativethrombotic risk could be reduced by the use of these heparins from 50 to60% to about 15 to 30% (J. Harenberg, S. Haas, and K. H. Breddin,“Prophylaxe der venösen Thrombose” in Müller-Berghaus, G. Pötsch (Ed.),“Hämostaseologie”, Springer Verlag, Berlin-Heidelberg, pages 564 to 580,1998). The achieved reduction of the peri- and post-operative thromboticrisk to 15 to 30% shows that there is a need for a further reduction ofthe thrombotic risk, and that is why research seeks to develop improvedalternatives.

The development of hirudine, a direct thrombin inhibitor, provided someimprovement because a more extensive protection became achievable.However, the use of hirudine is restricted to special problem cases,e.g., heparin intolerance, treatment of heparin-induced thrombocytopenia(HIT), because an antidote to heparin does not exist and thus there is arisk of uncontrollable bleedings.

Despite all the differences between the individual preparations, LMWheparins are rated clinically equivalent and routinely combined in metaanalyses. However, they are superior to UFH in orthopedics or insurgical hip joint replacement only with respect to effectiveness, butthere are no differences in tolerability. In the predominant applicationfield of general surgery, LWMH and standard heparin prove to beconstantly equivalent. This is true at least for the main criteria ofantithrombotic protection and tendency to bleeding.

The equivalence of the individual LMW heparins in clinics is in contrastto the differences in average molecular weight, molecular weightspectrum, the percent anti-FXa and anti-FIIa activities and theinfluence on APTT, a coagulation time which indicates the inhibition ofendogenous thrombin activity (by heparin). The list of differencesfurther comprises pro-fibrinolytic activity, release of TFPI (tissuefactor pathway inhibitor), influence on platelet function, etc.

To parallel the laboratory-medical variety of the individual products,there is no possibility of a superior clinical control of effectivenesswhich could determine the development of a new heparin.

To conclude, therefore, there are no indications of how to optimizeheparins with respect to therapeutic width as a ratio of effectivenessand tolerability.

Therefore, it has been the object of the present invention to provide anactive substance having anti-coagulant activity and thus to enrich theprior art by at least one further active substance, which activesubstance is to overcome at least part of the drawbacks known from theprior art.

Further, it has been an object of the present invention to provide a usefor the active substance according to the invention.

SUMMARY OF THE INVENTION

The present object is achieved by the use of a heparin having an averagemolecular weight ranging from 10 to 11.5 kd for the preparation of amedicament for the prophylaxis and therapy of thrombotic processes.Preferably the heparin has an average molecular weight of 10.5 kd.

Surprisingly, it has now been found that heparin having an averagemolecular weight of from 10 to 11.5 kd, more preferably heparin havingan average molecular weight of 10.5 kd, shows a range of activitieswhich is clearly different from that of UFH and LMWH.

In principle, due to the suitability of LMWH and UFH, one might haveexpected that the heparin according to the invention, having a mediummolecular weight, shows a range of activities which approximatelycorresponds to an average of the ranges of activities of UFH and LMWH.

Therefore, the present invention satisfies a long-standing need forimprovement of the prophylaxis and therapy of thrombo-embolic processes,especially also due to the fact that, while effectiveness is increased,there is no increased tendency to bleeding as compared to the knownheparins.

The evaluation of the heparin according to the invention was effected bya comparison with Enoxaparin, the internationally leading LMWH. SinceLMWH are generally considered to be at least as effective and tolerableas UFH, the results obtained from a comparison between the heparinaccording to the invention and Enoxaparin can also be extended to UFH insome way.

For further illustration, the invention will be explained in more detailin the following in the form of examples and comparisons with theheparins known from the prior art, the following specifications beingintended exclusively for further illustration of the invention and notfor its limitation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representation of the molecular weight distribution ofthe heparin according to the invention (MMWH), Enoxaparin (LMWH) andLiquemin® (UFH) in a GPC elution profile.

FIG. 2 a shows a representation of the respective APTT activity of theheparin according to the invention (MMWH), Enoxaparin (LMWH) andLiquemin® (UFH).

FIG. 2 b shows a representation of the anti-factor-Xa activities of thethree heparins.

FIG. 2 c shows a representation of the anti-thrombin activities of thethree heparins.

FIG. 3 a shows a representation of the AUC_(0-24h) values of the threeheparins.

FIG. 3 b shows a representation of the anti-FXa activities of the threeheparins which corresponds to that of FIG. 3 a.

FIG. 3 c shows a representation of the anti-FIIa activities of the threeheparins examined which corresponds to those of FIGS. 3 a and 3 b.

FIGS. 4 a to 4 c show a representation of the activities per unit dosein accordance with FIGS. 3 a to c of the three heparins examined.

FIG. 5 a shows a time-dependent representation of the concentration oftotal TFPI antigen respectively upon administration of a heparin (solidline: MMWH; dotted line: LMWH (Enoxaparin); dashed line: UFH(Liquemin®)).

FIG. 5 b shows a representation according to FIG. 5 a for the free TFPIantigen.

FIGS. 6 a to 6 d show a representation of the AUC₀₋₂₄ values for thethree heparins examined, calculated from the release of total TFPIantigen and free TFPI antigen.

FIG. 7 shows a comparison of the antithrombotic activity of Enoxaparinand that of the medium molecular weight heparin according to theinvention in an animal experiment (rabbit).

FIG. 8 shows a comparison of the duration of bleeding uponadministration of Enoxaparin and the medium molecular weight heparinaccording to the invention in an animal model (rabbit).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The heparin according to the invention was prepared by controlleddepolymerization and further processed by molecular filter techniques.The thus obtained heparin had the following HPLC profile:

number average molecular weight 10,513 weight average molecular weight10,819 Z average 11,233 viscosity average 10,819 Z + 1 average 11,815intrinsic viscosity 0.0000 peak molecular weight 10,236 dispersity1.029066 Z average/weight average 1.038271 Z + 1 average/weight average1.092039

For further characterization and for demonstrating the superiorproperties of the heparin according to the invention, the heparinaccording to the invention was compared with an unfractionated heparin(Liquemin®) and a low molecular weight heparin (Enoxaparin).

The medium molecular weight heparin according to the invention is alsoreferred to as “MMWH” in this description, Enoxaparin as arepresentative of the low molecular weight heparins is referred to as“LMWH”, and Liquemin® as a representative of the unfractionated heparinsis referred to as “UFH”.

With respect to their molecular weights and molecular weightdistributions, the heparin according to the invention, UFH and LMWH areclearly different, as can be seen from FIG. 1. From FIG. 1, whichrepresents a GPC elution profile of the three mentioned heparins, it canbe clearly seen that the UFH has an average molecular weight of 13.0 kdand at the same time a very broad molecular weight distribution. TheLMWH has an average molecular weight of 4.5 kd and a clearly narrowermolecular weight distribution. The heparin according to the invention,in contrast, has an average molecular weight of 10.5 kd, and it also hasthe narrowest molecular weight distribution of the three differentheparins.

The narrow molecular weight distribution of the heparin according to theinvention is of particular importance since it causes the unique andsurprising range of pharmacological activities of the heparin accordingto the present invention.

Substantial and unexpected differences are seen from a comparison of thein-vitro activities of the three heparins. Use is made of theinternationally standardized measuring system for anti-Xa activity inwhich the anti-Xa activity is based on 1 mg of the active substance. Forcomparison, measurements of anti-IIa activity were also performed. Theresults of this study are stated in the following Table 2.

TABLE 2 a-Xa a-Xa/a-IIa a-IIa/a-Xa IU/mg a-IIa IU/mg ratio ratio MMWH174.9¹ 170.0¹ 1.03 0.97 Liquemin ® (UFH) 159.0² 159.0² 1.00 1.00Enoxaparin (LMWH) 100.0²  26.3² 3.80 0.26 ¹determined according to the1st international standard for LMWH ²determined according to the 4thinternational standard for UFH

It is especially surprising that the heparin according to the invention(MMWH) shows a significant increase in activity as compared to the twocomparative active substances, which respectively represent a UFH and anLMWH, with respect to both anti-Xa activity and anti-IIa activity. Thiswas by no means to be expected. At best, one could have assumed that theanti-factor-Xa and anti-thrombin activities of the heparin according tothe invention would hold a position midway between those of UFH andLMWH.

A randomized multiple cross-over double-blind study was performed on 16healthy male subjects (age: 18–32 years, weight: 64–98 kg) for thepharmacokinetic and hemostasiological characterization of the heparinaccording to the invention (MMWH) as compared to Liquemin® (UFH) andEnoxaparin (LMWH). Each of the subjects-received one of the threeheparins as a single dose of 9000 anti-Xa IU within a one-week period.Blood was removed prior to each injection and after 0.5, 1, 2, 3, 4, 5,8, 10 and 24 hours from each injection, and buffered with citrate. Thefirst two milliliters of blood was discarded, and the plasma wasobtained according to general directions (I. Witt, H. Beeser and G.Müller-Berghaus, “Minimalanforderungen zur Gewinnung von Citratplasmafür hämostasiologische Analysen”, Lab. Med., pages 143–145, 1995). Thesamples obtained were divided into aliquots and immediately thereafterfrozen with liquid nitrogen and stored at −70° C. until the measurementswere performed. The ex-vivo studies which were then performed on theblood samples were subject to the conditions described in the following.

It is to be noted that the results presented hereinafter are the resultsof the in-vivo effects of the heparin according to the invention (MMWH)as compared to UFH and LMWH, which were examined ex vivo.

The anticoagulant activity of the plasma samples was established bydetermining the coagulation time according to an APTT (activated partialthrombin time) test (APTT-Micro Kieselgur, Instrumentation Laboratory),in which the inhibition of factor Xa and of the thrombin activity wasdetermined on an ACL 3000 model (Instrumentation Laboratory). Thisexamination includes the determination of a coagulation parameter whichis generally relied on for determining anticoagulant effects. Two valueswere generally determined. When the deviation was more than 3%, themeasurement was repeated. The validity was determined by controlexperiments performed in parallel on a comparative plasma, the 4thinternational standard for UFH and the 1st international standard forLMWH.

For the determination of the anti-Xa and anti-IIa activities, the plasmawas diluted with buffers for heparin determination (Chromogenix) andsupplemented with human AT (Chromogenix). After incubation with factorXa or thrombin (Chromogenix), the remaining activity was respectivelymeasured using the reaction of the chromogenic substrates S2222 andS2238.

The total and free TFPI antigen concentrations in the plasma sampleswere measured using the specific ELISAs “Asserachrom® Free TPFI” and“Asserachrom® Total TPFI” (Diagnostica Stago). ELISA plates coated withF(ab′)₂ fragments of a TFPI-specific antibody (TT4E2) were used for theTFPI measurement. The monoclonal peroxidase-coupled detector antibodieswere specific for either total TFPI (2C6) of free TFPI (H65).

The curves obtained from the studies for the prolongation of coagulationtime, inhibition of factor Xa and thrombin (factor IIa), and the totaland free TFPI concentrations in the individual subjects were used fordetermining the following pharmacokinetic parameters:

AUC_(0–24 h) [s x h, % inh. x h or (ng/ml) x h] = area under the curvec_(max) [s, % h inh. or ng/ml] = activity maximum (pro- longation ofcoagulation time, inhibition or concentration) t_(max) [min] = time ofactivity maximum t_(½,invasion) [min] = invasion half-lifet_(½,elimination) [min] = elimination half-life

The values for c_(max) and t_(max) were directly determined from theindividual curves. AUC was calculated between t=0 h and t=24 h using thetrapezoidal rule.

In addition to a base value of 0, a precondition of the determination oft_(1/2,invasion) and t_(1/2,elimination) is that the invasion andelimination have an exponential time course. To verify theseassumptions, a biexponential function was fitted to the measured data(Bateman function, extended by a log term). A comparison between thefitted and measured curves showed a very good agreement in most cases,so that the kinetics of heparin and TFPI can be described well by theBateman function. The values of t_(1/2,invasion) and t_(1/2,elimination)were calculated from the constants of the Bateman functions for invasionand elimination (k_(inv), K_(el)). All values were determined as meanvalues ± standard deviation. A comparison by pairs of all parametersbetween the three heparins was performed using a significance level (a)of 0.05. Differences were considered statistically significant if p wassmaller than 0.05. The mean values of the pharmacokinetic parameterswere subsequently subjected to a general linear model of varianceanalysis (null hypothesis: there is no difference respectively betweenthe heparin according to the invention and Liquemin® or Enoxaparin).

As a result, it is to be noted that the curves of the effects of thethree different heparins show clear and significant differences in thevarious studies, as can be seen from FIGS. 2 a to c.

It becomes clear therefrom that the heparin according to the inventionshows the highest activity in the APTT test and the anti-FIIa test andis surprisingly different from the other two heparins especially in itsanti-FIIa activity. Only the anti-FXa assay shows an expected result.Here, the heparin according to the invention holds a position aboutmidway between those of Enoxaparin and Liquemin® as examples of an LMWHand a UFH.

The different anticoagulant potentials of the three heparins examined isalso manifested in the AUC_(0-24h) data of the APTT test and theanti-FXa and anti-FIIa assays, which are represented in FIGS. 3 a to crespectively for the three heparins examined. The values result from theeffect of the respective heparin in the individual tests, i.e.,prolongation of coagulation time and the respective inhibition of factorXa and thrombin. It is found that the three heparins are significantlydifferent not only in their pharmacokinetics, but also in theirpharmacodynamics (e.g., the order of magnitude of anti-FXa activity ascompared to that of anti-FIIa activity).

What is striking is the fact that the activity of the heparin accordingto the invention as compared to Liquemin® is up to twofold respectivelyin all three assays. It can be concluded therefrom that thebioavailability of the heparin according to the invention, which doesnot contain the high molecular weight molecule typical of unfractionatedheparins, is significantly higher upon subcutaneous administration. Onthe other hand, a comparison of the heparin according to the inventionwith Enoxaparin shows that the heparin according to the invention doesnot belong to the low molecular weight heparins. While theanti-factor-Xa activity of Enoxaparin is more than twice that of theheparin according to the invention, its anti-thrombin activity is onlyabout 66% of that of the heparin according to the invention. It can beconcluded therefrom that the bioavailability of Enoxaparin is actuallyhigher, but that the thrombin-inhibiting activity, which is critical toanticoagulant activity, is significantly less pronounced than that ofthe heparin according to the invention.

The fact that the differences just described above in the in-vivoactivities of Enoxaparin and the heparin according to the inventionexist and are caused by the different bioavailabilities can be proven bya comparison with the already mentioned in-vitro activities of the threeheparins which are stated in Table 1. Here, the situation is completelydifferent, the heparin according to the invention showing the highestactivity with respect to both anti-FXa activity and anti-thrombinactivity. This difference only permits the conclusion that the influenceon the result of the studies from subcutaneous administration of thethree heparins to different test subjects is based on an alteredavailability of the active substances in the biological system, i.e.,bioavailability, in contrast to the in-vitro activities.

However, the values given in FIGS. 3 a to 3 b do not represent themass-specific activity of every single heparin, since the specificactivities of the three heparins are different, and 9000 IU of anti-FXaeach of the heparin according to the invention, Liquemin® and Enoxaparinhad been injected into the individual test subjects. This is also thereason why the values for AUC_(0-24h) were calculated per unit dose,i.e., the value for AUC_(0-24h)/mg of the respectively administeredheparin. The respectively administered doses were 51.5 mg of the heparinaccording to the invention, 56.5 mg of Liquemin® and 90 mg ofEnoxaparin. A comparison of the mass-specific AUC values, represented inFIGS. 4 a to 4 c, clearly shows that the heparin according to theinvention does not only have a higher bioavailability than that ofLiquemin®, but also an essentially higher potential as compared toEnoxaparin.

When the c_(max) values of the three heparins examined, which are basedon a prolongation of coagulation time and inhibition, are compared, itcan be seen that the values for the heparin according to the invention,such as the AUC_(0-24h) values, are higher than those of Liquemin®.However, the values were higher by at most 50% (anti-FXa activity),whereas the corresponding AUC_(0-24h) value was twice as high. Thisdiscrepancy is based on differences between the heparin according to theinvention and Liquemin® in some pharmacokinetic parameters. In addition,the superiority of the heparin according to the invention overLiquemin®, which is more pronounced for the AUC_(0-24h) value than forthe c_(max) value, may be of advantage because in the reverse case therisk of bleedings might be higher. By reducing the dose, c_(max) can bereduced while a higher AUC_(0-24h) value than that of Liquemin® ismaintained.

Similarly, the discrepancy between the c_(max) of Enoxaparin andLiquemin® in the anti-Xa and anti-thrombin assay is also lower than thatbetween the AUC_(0-24h) values. Despite its higher AUC_(0-24h) value,the c_(max) value of Enoxaparin is even 25% lower than that of Liquemin®in the APTT test. A comparison of the pharmacokinetic parametersc_(max), t_(1/2,invasion) [min] and t_(1/2,elimination) [min] shows thatthe heparin according to the invention is comparable to neither UFH norLMWH. Depending on the parameter, the heparin according to the inventionis clearly distinct from either both Liquemin® and Enoxaparin (e.g.,t_(1/2,invasion)) or only Liquemin® (e.g., t_(max)) or only Enoxaparin(e.g., t_(1/2,elimination)).

With respect to the time of the activity maximum, it may be set forthmerely that the heparin according to the invention has a t_(max) valuewhich is about the same as that of Enoxaparin and is only slightlydifferent from that of Liquemin®. In the three different assays, nosignificant differences could be observed for the three heparinsexamined.

The value for t_(1/2,invasion) is significantly longer for the heparinaccording to the invention than that for Liquemin® in the APTT test andin the anti-FXa assay, but it is not significantly longer in theanti-FIIa assay. In addition, this time is longer than that ofEnoxaparin in these two assays. However, in the anti-FIIa assay,Enoxaparin has the highest t_(1/2,invasion) value, which is significant.In contrast to other time-related values, the value fort_(1/2,elimination) of the heparin according to the invention is notsignificantly different from that of Liquemin®. The t_(1/2,elimination)value of Enoxaparin is twice as high than that of the heparin accordingto the invention and that of Liquemin in the APTT test and in theanti-FXa assay. However, in the anti-FIIa assay, Enoxaparin has thelowest value for t_(1/2,elimination). Therefore, the anti-FIIa activityof the heparin according to the invention is eliminated slowest, whichis to be considered an advantage over the other two heparins due to theimportance of anti-thrombin activity to the anticoagulant activity.

The above discussed values are summarized in the following in Table 3for the three heparins examined in the three assays.

TABLE 3 APTT activity anti-FXa activity anti-FIIa activity mean stand.mean stand. mean stand. value dev. relative p value value dev. relativep value value dev. relative p value AUC_(0–24 h) [s · h]² AUC_(0–24 h)[% inhibition · h]³ AUC_(0–24 h) [% inhibition · h] MMWH 154 66 1.45 439109 2.00 544 119 1.77 UFH 104 65 1.00 0.0001 219 80 1.00 0.0001 308 1151.00 0.0001 LMWH 126 55 1.21 0.0216 1028 114 4.69 0.0001 358 90 1.160.0001 AUC/dose AUC/dose [s · h/mg]³ AUC/dose [% inhibition · h/mg] [%inhibition · h/mg]³ MMWH 2.99 1.28 1.63 8.52 2.21 2.20 10.56 2.31 1.94UFH 1.84 1.43 1.00 n.d. 3.87 1.42 1.00 n.d. 5.44 2.04 1.00 n.d. LMWH1.40 0.616 0.76 n.d. 11.42 1.27 2.95 n.d. 3.97 1.00 0.73 n.d. c_(max)[s]² c_(max) [% inhibition] c_(max) [% inhibition] MMWH 22.7 10.7 1.3046.7 11.5 1.49 55.5 14.3 1.36 UFH 17.4 12.7 1.00 0.0001 31.4 12.2 1.000.0001 41.0 14.8 1.00 0.0001 LMWH 13.0 4.5 0.75 0.0216 77.2 7.9 2.460.0001 41.3 12.5 1.01 0.0001 t_(max) [min] t_(max) [min] t_(max) [min]MMWH 233 82 1.31 218 43 1.28 221 46 1.20 UFH 178 73 1.00 0.0175 171 461.00 0.0036 184 46 1.00 0.0081 LMWH 240 49 1.35 0.7353 221 36 1.300.8070 248 37 1.35 0.0588 t_(½,invasion) [min] t_(½,invasion) [min]t_(½,invasion) [min] MMWH 105 17 1.57 116 24 1.29 99 36 1.18 UFH 67 371.00 0.0003 91 26 1.00 0.0057 84 34 1.00 0.0637 LMWH 82 28 1.23 0.022489 24 0.98 0.0037 122 22 1.45 0.0082 t_(½,elimination) [min]t_(½,elimination) [min] t_(½,elimination) [min] MMWH 129 68 0.84 137 281.19 171 59 1.22 UFH 153 107 1.00 0.7753 115 40 1.00 0.2378 140 64 1.000.0572 LMWH 225 126 2.18 0.0187 252 83 2.19 0.0001 134 29 0.96 0.0239 ¹p< 0.05; significant; ²base of calculation: prolongation of coagulationtime; ³administered doses: 5.5 mg of MMWH, 56.5 mg of Liquemin ® (UFH),90.0 mg of Enoxaparin (LMWH)

Upon consideration and comparison of the total TFPI antigenconcentrations and the concentrations of the free TFPI antigen afteradministration of the three different heparins, it is found that theconcentration of the total TFPI antigen has increased to 2.0-fold to2.5-fold of the initial value after one hour, and that the concentrationof the free TFPI antigen has increased to 6.7-fold to 7.9-fold of theinitial concentration. From FIGS. 5 a and 5 b, it can be seen that themaximum values are found after 1.5 to 2 hours, so that t_(max) issignificantly shorter than it was in the three assays performedpreviously. This shows that the heparin-induced TFPI release does notcorrelate with these traditional coagulation parameters.

Further, it can be seen from FIG. 5 a and FIG. 5 b that the activity ofthe heparin according to the invention is clearly different from thoseof Liquemin® and Enoxaparin. After administration of the heparinaccording to the invention, both values examined in this connectionremain longer on a high level as compared with the administration of oneof the comparative heparins.

This effect of the heparin according to the invention is also verysurprising and of particular importance to the present invention, sinceespecially the free TFPI antigen is considered to play an important rolein coagulation.

A comparison of the corresponding AUC_(0-24h) values on the basis of theconcentrations of released TFPI (for total TFPI antigen and free TFPIantigen) makes clear that the heparin according to the invention andEnoxaparin increase the plasma concentrations of the total TFPI antigenabout in the same way and are clearly superior to Liquemin®, as can beseen from FIG. 6 a.

However, since the concentration of the free TFPI antigen is theparameter which is critical to coagulation where the contribution ofTFPI to the activity of heparin is concerned, the AUC_(0-24h) values forthe concentration of the free TFPI antigen would have to be compared.From FIG. 6 b, it can be seen that the heparin according to theinvention is found the most effective.

Now, when the AUC_(0-24h) values per unit dose are compared on the basisof the concentrations of released TFPI, the result shown in FIGS. 6 cand 6 d is obtained.

Both the concentration of total TFPI antigen and the concentration offree TFPI antigen are highest for the heparin according to theinvention. It is to be noted that the heparin according to the inventionis clearly superior to the known heparins to an extremely andsurprisingly high extent for two parameters critical to coagulation,i.e., anti-thrombin activity and release of free TFPI antigen.

Finally, it is to be mentioned that these results are confirmed by acomparison of the heparin according to the invention with Enoxaparin inan animal experiment. This animal experiment was performed on rabbitsand clearly showed the better therapeutic effect of the heparinaccording to the invention in the prevention and treatment ofthrombo-embolic processes as compared to low molecular weight heparins.In addition, a lower risk was found with respect to the occurrence ofbleedings after the administration of the heparin according to theinvention as compared to Enoxaparin.

Further, due to the examination results described above, it is preferredto employ the medium molecular weight heparin according to the inventionalso for the therapy of acute myocardial infarction and unstable angina,and for the inhibition of coagulation in extracorporeal circulations.

It is to be pointed out that the surprising properties of the mediummolecular weight heparin according to the invention are shown, inparticular, in the APTT test, which is a basic measure in heparintherapy. The coagulation time in the APTT test is significantlyprolonged as compared to the examined heparins Enoxaparin and Liquemin®.The particularly high milligram activity of the heparin according to theinvention, which is also completely surprising, is also of importance.Together with a significantly decreased risk of bleeding, which in ananimal experiment impressively demonstrates the superiority of themedium molecular weight heparin according to the invention, completelynew perspectives in heparin therapy are opened by the present invention.

With respect to activity and tolerability, the medium molecular weightheparin according to the invention was also compared to a low molecularweight heparin, i.e., Enoxaparin, in an animal experiment. This was donewithin the scope of a modified thrombosis model according to S. Wessler(“rabbit stasis model”, “thrombosis in the presence of vascular stasis”,Am. J. Med., 33: 649, 1959), wherein the experimentally inducedthrombosis results from a combination of hypercoagulability andvenostasis.

Two methods proven many times and well-established which served fordetermining the tendency to bleeding were the so-called “rabbit bloodloss model” (counting of erythrocytes in the rinsing fluid after adefined injury) and the “rabbit template bleeding time” in which thebleeding time is determined (J. Fareed, J. M. Walenga et al., Studies onthe antithrombotic effects and pharmacokinetics of heparin fractions andfragments, Sem. Thromb. Hemost. 11, 56–74, 1985).

FIGS. 7 and 8 prove the impressive consistent increase of the“therapeutic index” of the medium molecular weight heparin according tothe invention as compared to LMWH, which is based not only on theenhanced antithrombotic activity, but also particularly on thesignificant reduction of the tendency to bleeding.

FIG. 7 again shows a comparison of the antithrombotic activity uponintravenous administration of Enoxaparin used as the low molecularweight heparin with that of the medium molecular weight heparin of thepresent invention. From the Figure, it can be seen that the coagulationvalue, which is a measure of the number and size of the blood clotsformed, is significantly lower for the heparin according to theinvention as compared to Enoxaparin. Saline was respectively used as azero control. It was also found that clotting is completely suppressedalso by Enoxaparin only at an elevated dose, which is presently at 50units/kg of body weight.

FIG. 8 shows a comparison of the bleeding time upon intravenousadministration of Enoxaparin, medium molecular weight heparin, andsaline as a zero control or standard. It was found just at the higherdoses that the medium molecular weight heparin according to theinvention has a substantially higher potential as compared toEnoxaparin, since its absolutely lower tendency to bleeding becomesclear from the animal experiment.

Now, when these results are summarized, a so-called therapeutic indexwith respect to the low molecular weight heparin Enoxaparin can beestablished for the medium molecular weight heparin according to theinvention. Thus, the data for effectiveness, i.e., antithromboticactivity, and the data for the tendency to bleeding are evaluated andcorrelated by means of variance analysis and linear regression. Theresult shows a therapeutic index of 2.24 for the medium molecular weightheparin according to the invention with respect to Enoxaparin. In otherwords, the medium molecular weight heparin according to the inventionhas an application value which is 2.24 times as high as that ofEnoxaparin. These results are completely surprising to the skilledperson and enable a significant improvement of heparin therapy about 60years after the first use of unfractionated heparins and about 20 yearsafter the first use of the low molecular weight heparins. Therefore, thepresent invention satisfies a long-standing need in the art.

The present invention provides a heparin which is explicitly better thanthose of the prior art and is characterized by a higher therapeuticindex which is based on a more favorable ratio of effectiveness andtolerability. For clinical use, a greater reach in prophylaxis andtherapy can be prognosticated: The medium molecular weight heparinaccording to the invention will be capable of being relatively higherdosed as compared to the known heparins and thus contribute to animprovement in the managing of thrombo-embolic diseases which is not yetexactly predictable.

This is unexpected for the particular reason that is cannot be derivedfrom clinical parameters.

1. A process for treatment of thrombo-embolic processes, comprising:administering an effective amount of a medium molecular weight heparin(MMWH) pharmaceutical preparation to a subject in need of treatment ofthrombo-embolic processes, wherein the effective amount is sufficient totreat the thrombo-embolic processes and is characterized as having thehigher therapeutic index of MMWH relative to low molecular weightheparin (LMWH) and unfractionated (UFH) and the MMWH has an averagemolecular weight ranging from 10 to 11.5 kDa.
 2. The process accordingto claim 1, wherein the MMWH has an average molecular weight of 10.5kDa.
 3. The process of claim 1 wherein the thrombo-embolic processesinclude those in acute myocardial infarction, unstable angina andcoagulation in extracorporeal circulations.
 4. The process of claim 1wherein the MMWH has a a-Xa/a-IIa ratio of 1.03.